Dopamine (DA) is an essential neurotransmitter and plays a major role in the rewarding effects of psychostimulants, including cocaine and amphetamine. A primary component of DA neurotransmission that determines the lifetime of DA at synapses is the re-uptake of the transmitter back into nerve terminals by the DA transporter (DAT). Importantly, many psychostimulants produce their reinforcing effects primarily by altering the function of DAT. Therefore, it becomes necessary to understand the regulatory mechanisms associated with DAT function in order to assess their role in mediating the effects of pyschostimulants. Recently, we and others have identified several DAT protein interactions, suggesting that synaptic distribution, functional properties, and molecular actions of psychostimulants at DAT can be regulated via associated proteins. Specifically, we identified the synaptic vesicle protein synaptogyrin-3 (SG3) as a DAT interacting protein using a proteomic approach. These studies have produced ample data demonstrating a physical and functional interaction between the amino terminus of DAT (DATN) and SG3 in vitro. We hypothesize that the DAT/SG3 interaction facilitates synaptic vesicle docking at the plasma membrane near DAT to provide efficient loading of the vesicles with extracellular dopamine during the reuptake process. Importantly, this interaction might mediate some actions of psychostimulants. To date, the impact of protein-protein interactions on DA homeostasis or the effects of psychostimulants has not been examined in vivo. In order to provide a mode of investigation for the role of DAT protein-protein interactions in the regulation of DA homeostasis in vivo, we propose an explorative approach that will combine two contemporary methodologies. Fast scan cyclic voltammetry (FSCV), has been used by many in conjunction with electrical stimulation and/or pharmacological manipulation to obtain information about temporal changes and concentration of catecholamines in the central nervous system. The second contemporary technique we will employ involves the use of TAT-conjugated interfering peptides to effectively deliver the DAT binding domain of SG3 to the brain. Our group has begun to use these two approaches together with the goal of discerning whether disrupting the DAT/SG3 interaction impacts DA neurotransmission. Indeed, preliminary data suggests that administration of the DAT-N fused to TAT disrupts the interaction between SG3 and DAT and has profound adverse consequences on in vivo striatal DA neurotransmission. We propose to examine systematically the functional consequences of disrupting the DAT/SG3 protein-protein interaction on DA neurotransmission and to evaluate the effects of this interaction on psychostimulant response. The studies described in this proposal are expected to support the goal of developing novel therapies for drug addiction. [unreadable] [unreadable] [unreadable]